The so-called “triangulating” autofocus device as known from U.S. Pat. No. 5,136,149 B1 belongs to the aforementioned category of position-sensing autofocus devices. DE 195 37 376 A1 refers to the autofocus principle described in the aforesaid US patent document as a “triangulating” autofocus principle. Autofocus devices of this kind, also called “autofocus scanning” units, use an autofocus measuring beam, incident obliquely or in raking fashion onto a specimen, as an autofocus beam path; this measuring beam is focused by the objective of the microscope onto the specimen, where a measurement pattern, generally in the form of a spot or slit, is produced. This autofocus principle requires a mirror-reflective or evenly reflective autofocus interface. After reflection, the measuring beam passes again through the microscope objective and can then be diverted into a position-sensitive autofocus detector. This detector detects the lateral shift of the measuring beam, the reason being that if the distance between the objective and the autofocus interface (“focus hold”) changes, a lateral shift of the measuring beam occurs at the detector, with the result that a signal modified with respect to the focus position can be generated. The degree of defocusing can thereby be measured, and then compensated for using suitable means. One compensation possibility is a motor that moves the objective lens correspondingly in order to cancel out the change that occurred in the distance between objective and autofocus interface. Further details regarding the configuration and manner of operation of a triangulating autofocus device may be gathered from the previously mentioned documents U.S. Pat. No. 5,136,149 B1 and DE 195 37 376 A1.
DE 32 19 503 A1 discloses a similar autofocusing device for optical equipment, in particular incident-light microscopes. With this apparatus a laser autofocusing arrangement is provided which generates a measuring beam bundle one of whose halves is blocked out by means of an optical component. The measuring beam bundle, limited to half its cross section, is coupled as an autofocus measuring beam into the illumination beam path of the incident-light microscope, which in turn is incident via the objective pupil and the objective onto a specimen. The half-blocked measuring beam—preferably pulsed laser light in the IR region—thereby generates a measurement spot (which does not interfere with microscopic observation) on the specimen for autofocusing. In the event of defocusing, this measurement spot “migrates” over the surface of the specimen. After reflection at the specimen surface, the remitted autofocus measurement (half-) beam proceeds back to the optical component (deflection prism) and from there to a detector that can be made up substantially of a differential diode (two diodes). When the system is optimally focused, the image of the measurement spot is located in an exactly symmetrical position with reference to the two diodes of the detector. In the event of defocusing, the image of the measurement spot migrates out of the central position toward one of the two diodes, depending on the defocusing direction. To a first approximation, the magnitude of the displacement of the measurement spot on the differential diode is proportional to the magnitude of the defocusing. The apparatus allows the detected defocusing to be canceled out by corresponding counter-control of the objective and/or of the specimen stage in the Z direction (direction of the optical axis).
An autofocus system having a similar measurement principle is also known from US 2004/0113043 A1. The correlation between the detected signal and the actual focus position is depicted graphically in this US document.
A similar autofocus system for an inverted microscope with transmitted illumination is known from U.S. Pat. No. 7,345,814 B2. To minimize flare, a polarizing beam splitter and a λ/4 plate are provided in the beam path of the autofocus apparatus.
DE 601 16 268 T2 also describes a variety of embodiments of an autofocus device for a high-throughput screening microscope, in which the displacement and shape of an autofocus measurement spot acquired by an autofocus detector are analyzed in order to determine therefrom the degree of defocusing and then to keep the focus constant during a screening procedure.
WO 2009/092555 A1 describes an autofocus method of the second category, namely image content analysis, for microscopes. Here a grating incorporated obliquely into the illumination beam path in the illuminated field plane is imaged onto a specimen as a focusing image, for autofocusing purposes, by means of an autofocusing optical system, deflection mirrors, and the microscope objective. The grating can be embodied, for example, as a groove grating. In the arrangement described therein, the focusing image plane in which the grating focusing image is located encloses a specific angle with the focal plane of the imaging optical system of the microscope. When focusing is optimal, the focusing image acquired by a CCD camera of the microscope is sharpest along the intersection line of the two aforesaid planes that lies at the center of the image, and the sharpness decreases outward in both directions. The same is true of the contrast of this image. If the specimen becomes defocused, said intersection line migrates in a lateral direction, and the location of the best image of the grating focusing image shifts correspondingly. Defocusing of the specimen (i.e. a deviation in the Z direction) results in a lateral displacement of the location of the sharpest image on the CCD detector. This document proposes to derive an intensity profile from the focusing image acquired by the CCD camera, and to ascertain from that profile intensities that can be plotted as a function of distance Z from the focal plane. An intensity maximum is thus located at the optimal focus location. In addition, a contrast profile can be derived from the intensity profile by way of a convolution operation. Once again, a contrast maximum is located at the point of optimal focus. A control unit calculates the defocusing on the basis of the profiles, and compensates for it by correspondingly displacing the specimen stage of the microscope.
In the case of the above-described triangulating autofocus devices, the lateral displacement of the center point of the measurement spot, the edge location of the measurement spot or slit, or a fitted profile is used as a signal from which the degree of defocusing is derived. In the case of the autofocus device just mentioned, a signal of this kind is obtained from the intensity or contrast of an imaged grating. The entire imaging path, from the light source (or a structure in the field diaphragm) through the objective and the specimen to the detector, is relevant to the signal that is to be evaluated. If components such as a light source, detector, or deflection elements shift as a result of thermal drift, this directly influences the signal. Errors in determining the defocusing are thereby caused, resulting in inaccurate autofocusing. It typically takes two hours and more for a corresponding configuration to be sufficiently thermally stable to allow precise long-duration experiments to be carried out.
In a method according to WO 2009/092555 A1 (see statements above), the interface between the coverslip and an aqueous sample embedding medium is also imaged onto the detector, the interface being imaged directly onto the CCD detector. Located in this interface are not only the cells that are intended to be observed, but also impurities, scratches, bubbles, and other disruptions that are overlaid directly onto the image and thus onto the signal that is derived, and consequently complicate or distort the evaluation.